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Doxorubicin (DOX) is a broad-spectrum, highly effective antitumor agent; however, its cardiotoxicity has greatly limited its use. Hydrogen sulfide (H2S) is an endogenous gaseous transmitter that exerts cardioprotective effects via the regulation of oxidative stress and apoptosis and maintenance of mitochondrial function, among other mechanisms. AP39 is a novel mitochondria-targeted H2S donor that, at appropriate concentrations, attenuates intracellular oxidative stress damage, maintains mitochondrial function, and ameliorates cardiomyocyte injury. In this study, DOX-induced cardiotoxicity models were established using H9c2 cells and Sprague-Dawley rats to evaluate the protective effect of AP39 and its mechanisms of action. Both in vivo and in vitro experiments showed that DOX induces oxidative stress injury, apoptosis, and mitochondrial damage in cardiomyocytes and decreases the expression of p-AMPK/AMPK and UCP2. All DOX-induced changes were attenuated by AP39 treatment. Furthermore, the protective effect of AP39 was significantly attenuated by the inhibition of AMPK and UCP2. The results suggest that AP39 ameliorates DOX-induced cardiotoxicity by regulating the expression of AMPK/UCP2., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The lymphatic network is pivotal for various physiological functions in the human body. Accumulated evidence supports the role of therapeutic lymphangiogenesis in the treatment of several pathologies. Endogenous gasotransmitter, hydrogen sulfide (H2S) has been extensively studied for its potential as a pro-angiogenic factor and vascular function modulator. However, the role of H2S in governing lymphatic vessel formation, and underlying molecular mechanisms are understudied. The present study was designed to investigate the effects of H2S donor sodium hydrogen sulfide (NaHS) on lymphatic vascularization and pro-angiogenic signaling pathways using both in vitro and in vivo approaches. In vitro dose-response experiments showed increased proliferation and tube formation by NaHS-treated human lymphatic endothelial cells (LECs) compared with control cells. Immunoblotting performed with LEC lysates prepared after time-course NaHS treatment demonstrated increased activation of ERK1/2, AKT and eNOS after 20 min of NaHS stimulation. Further, NaHS treatment induced nitric oxide production, reduced reactive oxygen species generation, and promoted cell cycle in LECs. Additional cell cycle analysis showed that NaHS treatment abrogates oxidized LDL-induced cell cycle arrest in LECs. The results of in vivo Matrigel plug assay revealed increased lymphatic vessel density in Matrigel plugs containing NaHS compared with control plugs, however, no significant differences in angiogenesis and immune cell infiltration were observed. Collectively, these findings suggest that H2S donor NaHS promotes lymphatic vessel formation both in vitro and in vivo and may be utilized to promote reparative lymphangiogenesis to alleviate lymphatic dysfunction-related disorders., Competing Interests: the authors disclose no competing interests., (Copyright: © 2023 Aithabathula et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Both hydrogen sulfide (H2S) and sulfur dioxide (SO2) are produced endogenously from the mammalian metabolic pathway of sulfur-containing amino acids and play important roles in several vascular diseases. However, their interaction during the control of vascular function has not been fully clear. Here, we investigated the potential role of H2S in SO2 production and vascular regulation in vivo and in vitro. Wistar rats were divided into the vehicle, SO2, DL-propargylglycine (PPG) + SO2, β-cyano-L-alanine (BCA) + SO2 and sodium hydrosulfide (NaHS) + SO2 groups. SO2 donor was administered with or without pre-administration of PPG, BCA or NaHS for 30 min after blood pressure was stabilized for 1 h, and then, the change in blood pressure was detected by catheterization via the common carotid artery. Rat plasma SO2 and H2S concentrations were measured by high performance liquid chromatography and sensitive sulfur electrode, respectively. The isolated aortic rings were prepared for the measurement of changes in vasorelaxation stimulated by SO2 after PPG, BCA or NaHS pre-incubation. Results showed that the intravenous injection of SO2 donors caused transient hypotension in rats compared with vehicle group. After PPG or BCA pretreatment, the plasma H2S content decreased but the SO2 content increased markedly, and the hypotensive effect of SO2 was significantly enhanced. Conversely, NaHS pretreatment upregulated the plasma H2S content but reduced SO2 content, and attenuated the hypotensive effect of SO2. After PPG or BCA pre-incubation, the vasorelaxation response to SO2 was enhanced significantly. While NaHS pre-administration weakened the SO2-induced relaxation in aortic rings. In conclusion, our in vivo and in vitro data indicate that H2S negatively controls the plasma content of SO2 and the vasorelaxant effect under physiological conditions., Competing Interests: The authors have declared that no competing interests exist.

Background: Since the start of organ transplantation, hypothermia-forced hypometabolism has been the cornerstone in organ preservation. Cold preservation showed to protect against ischemia, although post-transplant injury still occurs and further improvement in preservation techniques is needed. We hypothesize that hydrogen sulphide can be used as such a new preservation method, by inducing a reversible hypometabolic state in human sized kidneys during normothermic machine perfusion., Methods: Porcine kidneys were connected to an ex-vivo isolated, oxygen supplemented, normothermic blood perfusion set-up. Experimental kidneys (n = 5) received a 85mg NaHS infusion of 100 ppm and were compared to controls (n = 5). As a reflection of the cellular metabolism, oxygen consumption, mitochondrial activity and tissue ATP levels were measured. Kidney function was assessed by creatinine clearance and fractional excretion of sodium. To rule out potential structural and functional deterioration, kidneys were studied for biochemical markers and histology., Results: Hydrogen sulphide strongly decreased oxygen consumption by 61%, which was associated with a marked decrease in mitochondrial activity/function, without directly affecting ATP levels. Renal biological markers, renal function and histology did not change after hydrogen sulphide treatment., Conclusion: In conclusion, we showed that hydrogen sulphide can induce a controllable hypometabolic state in a human sized organ, without damaging the organ itself and could thereby be a promising therapeutic alternative for cold preservation under normothermic conditions in renal transplantation., Competing Interests: The authors have declared that no competing interests exist.

S-sulfhydration is a signalling pathway of hydrogen sulfide (H2S), which is suggested as an anti-atherogenic molecule that may protect against atherosclerosis. The identification of S-sulfhydrated proteins by proteomic approach could be a major step towards understanding the mechanisms of H2S in response to atherosclerosis. The present study studied targeted S-sulfhydrated proteins using the modified biotin switch method followed by matrix-assisted laser desorption/ionisation time of flight tandem mass spectrometry identification. The results showed that H2S can protect against atherosclerosis by reducing body weight gain and alleviating aortic plaque formation. In addition, H2S treatment can increase aortic protein S-sulfhydration. Seventy targeted S-sulfhydrated aortic proteins were identified, mainly involved in metabolism, stimulus response and biological regulation, as determined by gene ontology database analysis. H2S also induced S-sulfhydration of glutathione peroxidase 1 and further reduced lipid peroxidation and increased antioxidant defence in the aorta by prompting glutathione synthesis. Our data suggest that H2S is a cardiovascular-protective molecule that S-sulfhydrates a subset of proteins that are mainly responsible for lipid metabolism and exerts its cytoprotective effects to clear free radicals and inhibit oxidative stress through cysteine S-sulfhydration.

A hydroponic experiment was performed to investigate the stress responses and biochemical adaptations of four submerged macrophytes, Potamogeton crispus, Myriophyllum spicatum, Egeria densa, and Potamogeton oxyphyllus, to the combined exposure of hypoxia and hydrogen sulfide (H2S, provided by NaHS). The investigated plants were subjected to a control, hypoxia, 0.1mM NaHS, 0.5 mM NaHS, 0.1 mM NaHS+hypoxia and 0.5 mM NaHS+hypoxia conditions. All experimental plants grew optimally under control, hypoxic and NaHS conditions in comparison to that grown in the combined exposure of hypoxia and hydrogen sulfide. For P. crispus and M. spicatum, significant decreases of total chlorophyll and increases in oxidative stress (measured by hydrogen peroxide, H2O2, and malondialdehyde, MDA) were observed with exposure to both sulfide concentrations. However, the decrease in catalase (CAT) and ascorbate peroxidase (APX) from exposure to 0.5 mM NaHS suggests that the function of the protective enzymes reached their limit under these conditions. In contrast, for E. densa and P. oxyphyllus, the higher activities of the three antioxidative enzymes and their anaerobic respiration abilities (ADH activity) resulted in higher tolerance and susceptibility under high sulfide concentrations.

Although essential in critical care medicine, mechanical ventilation often results in ventilator-induced lung injury. Low concentrations of hydrogen sulfide have been proven to have anti-inflammatory and anti-oxidative effects in the lung. The aim of this study was to analyze the kinetic effects of pre- and posttreatment with hydrogen sulfide in order to prevent lung injury as well as inflammatory and oxidative stress upon mechanical ventilation. Mice were either non-ventilated or mechanically ventilated with a tidal volume of 12 ml/kg for 6 h. Pretreated mice inhaled hydrogen sulfide in low dose for 1, 3, or 5 h prior to mechanical ventilation. Posttreated mice were ventilated with air followed by ventilation with hydrogen sulfide in various combinations. In addition, mice were ventilated with air for 10 h, or with air for 5 h and subsequently with hydrogen sulfide for 5 h. Histology, interleukin-1β, neutrophil counts, and reactive oxygen species formation were examined in the lungs. Both pre-and posttreatment with hydrogen sulfide time-dependently reduced or even prevented edema formation, gross histological damage, neutrophil influx and reactive oxygen species production in the lung. These results were also observed in posttreatment, when the experimental time was extended and hydrogen sulfide administration started as late as after 5 h air ventilation. In conclusion, hydrogen sulfide exerts lung protection even when its application is limited to a short or delayed period. The observed lung protection is mediated by inhibition of inflammatory and oxidative signaling.

Little information is available describing the effects of exogenous H2S on the ABA pathway in the acquisition of drought tolerance in wheat. In this study, we investigated the physiological parameters, the transcription levels of several genes involved in the abscisic acid (ABA) metabolism pathway, and the ABA and H2S contents in wheat leaves and roots under drought stress in response to exogenous NaHS treatment. The results showed that pretreatment with NaHS significantly increased plant height and the leaf relative water content of seedlings under drought stress. Compared with drought stress treatment alone, H2S application increased antioxidant enzyme activities and reduced MDA and H2O2 contents in both leaves and roots. NaHS pretreatment increased the expression levels of ABA biosynthesis and ABA reactivation genes in leaves; whereas the expression levels of ABA biosynthesis and ABA catabolism genes were up-regulated in roots. These results indicated that ABA participates in drought tolerance induced by exogenous H2S, and that the responses in leaves and roots are different. The transcription levels of genes encoding ABA receptors were up-regulated in response to NaHS pretreatment under drought conditions in both leaves and roots. Correspondingly, the H2S contents in leaves and roots were increased by NaHS pretreatment, while the ABA contents of leaves and roots decreased. This implied that there is complex crosstalk between these two signal molecules, and that the alleviation of drought stress by H2S, at least in part, involves the ABA signaling pathway., Competing Interests: The authors have declared that no competing interests exist.

A major cause of preterm labor in pregnant women is intra-amniotic infection, which is mediated by an inflammatory process. Hydrogen sulfide (H2S), a gaseous transmitter, has been implicated to be involved in inflammatory responses. We sought to investigate whether H2S affects infectious preterm birth using the mouse model of lipopolysaccharides (LPS)-induced preterm birth. Administration of LPS at 0.4 mg/kg with two injections intraperitoneally (i.p.) on gestational day 14.5 induced preterm labor. LPS significantly increased leukocyte infiltration in uterus, stimulated the expression of pro-inflammatory cytokines interleukin 1β (IL-1β), IL-6, tumor necrosis factor α (TNF-α), CCL2 and CXCL15 in myometrium. Administration of NaHS (i.p.) delayed the onset of labor induced by LPS in a dose-dependent manner. NaHS prevented leukocyte infiltration into intrauterine tissues and inhibited the production of pro-inflammatory cytokines in myometrium and decreased the levels of these cytokines in maternal circulation. H2S also decreased LPS-activated extracellular signal-regulated kinase (ERK) 1/2/ nuclear factor (NF)-κB signaling pathways in myometrium. This study provides new in vivo evidence for the roles of H2S in attenuating inflammation, and a potential novel therapeutic strategy for infection-related preterm labor.

Hydrogen sulfide (H2S) has anti-fibrotic potential in lung, kidney and other organs. The exogenous H2S is released from sodium hydrosulfide (NaHS) and can influence the renal fibrosis by blocking the differentiation of quiescent renal fibroblasts to myofibroblasts. But whether H2S affects renal epithelial-to-mesenchymal transition (EMT) and the underlying mechanisms remain unknown. Our study is aimed at investigating the in vitro effects of H2S on transforming growth factor-β1 (TGF-β1)-induced EMT in renal tubular epithelial cells (HK-2 cells) and the associated mechanisms. The induced EMT is assessed by Western blotting analysis on the expressions of α-SMA, E-cadherin and fibronectin. HK-2 cells were treated with NaHS before incubating with TGF-β1 to investigate its effect on EMT and the related molecular mechanism. Results demonstrated that NaHS decreased the expression of α-SMA and fibronectin, and increased the expression of E-cadherin. NaHS reduced the expression of TGF-β receptor type I (TβR I) and TGF-β receptor type II (TβR II). In addition, NaHS attenuated TGF-β1-induced increase of β-catenin expression and ERK phosphorylation. Moreover, it inhibited the TGF-β1-induced nuclear translocation of ββ-catenin. These effects of NaHS on fibronectin, E-cadherin and TβR I were abolished by the ERK inhibitor U0126 or β-catenin inhibitor XAV939, or β-catenin siRNA interference. We get the conclusion that NaHS attenuated TGF-β1-induced EMT in HK-2 cells through both ERK-dependent and β-catenin-dependent pathways.

To identify novel susceptibility variants for osteoporosis in Korean postmenopausal women, we performed a genome-wide association analysis of 1180 nonsynonymous single nucleotide polymorphisms (nsSNPs) in 405 individuals with osteoporosis and 722 normal controls of the Korean Association Resource cohort. A logistic regression analysis revealed 72 nsSNPs that showed a significant association with osteoporosis (p<0.05). The top 10 nsSNPs showing the lowest p-values (p = 5.2×10-4-8.5×10-3) were further studied to investigate their effects at the protein level. Based on the results of an in silico prediction of the protein's functional effect based on amino acid alterations and a sequence conservation evaluation of the amino acid residues at the positions of the nsSNPs among orthologues, we selected one nsSNP in the SQRDL gene (rs1044032, SQRDL I264T) as a meaningful genetic variant associated with postmenopausal osteoporosis. To assess whether the SQRDL I264T variant played a functional role in the pathogenesis of osteoporosis, we examined the in vitro effect of the nsSNP on bone remodeling. Overexpression of the SQRDL I264T variant in the preosteoblast MC3T3-E1 cells significantly increased alkaline phosphatase activity, mineralization, and the mRNA expression of osteoblastogenesis markers, Runx2, Sp7, and Bglap genes, whereas the SQRDL wild type had no effect or a negative effect on osteoblast differentiation. Overexpression of the SQRDL I264T variant did not affect osteoclast differentiation of the primary-cultured monocytes. The known effects of hydrogen sulfide (H2S) on bone remodeling may explain the findings of the current study, which demonstrated the functional role of the H2S-catalyzing enzyme SQRDL I264T variant in osteoblast differentiation. In conclusion, the results of the statistical and experimental analyses indicate that the SQRDL I264T nsSNP may be a significant susceptibility variant for osteoporosis in Korean postmenopausal women that is involved in osteoblast differentiation.

The effect of hydrogen sulfide (H2S) on differentiation of 3T3L1-derived adipocytes was examined. Endogenous H2S was increased after 3T3L1 differentiation. The expression of the H2S-synthesising enzymes, cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), was increased in a time-dependent manner during 3T3L1 differentiation. Expression of genes associated with adipogenesis related genes including fatty acid binding protein 4 (FABP4/aP2), a key regulator of this process, was increased by GYY4137 (a slow-releasing H2S donor compound) and sodium hydrosulfide (NaHS, a classical H2S donor) but not by ZYJ1122 or time-expired NaHS. Furthermore expression of these genes were reduced by aminooxyacetic acid (AOAA, CBS inhibitor), DL-propargylglycine (PAG, CSE inhibitor) as well as by CSE small interference RNA (siCSE) and siCBS. The size and number of lipid droplets in mature adipocytes was significantly increased by both GYY4137 and NaHS, which also impaired the ability of CL316,243 (β3-agonist) to promote lipolysis in these cells. In contrast, AOAA and PAG had the opposite effect. Taken together, we show that the H2S-synthesising enzymes CBS, CSE and 3-MST are endogenously expressed during adipogenesis and that both endogenous and exogenous H2S modulate adipogenesis and adipocyte maturation.

Objective: To examine the hypothesis that hydrogen sulfide (H2S) regulates the colonic motility by modulating both L-type voltage-dependent calcium channels and large conductance Ca2+-activated K+ (BKCa) channels., Methods: Immunohistochemistry was performed on rat colonic samples to investigate the localization of the H2S-producing enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). The contractions of proximal colonic smooth muscle were studied in an organ bath system. The whole-cell patch-clamp technique was used to record both L-type calcium currents (ICa,L) and BKCa currents in colonic smooth muscle cells (SMCs) isolated from male Wistar rats., Results: Immunohistochemistry revealed the presence of CBS and CSE in mucosa, smooth muscle cells and myenteric neurons. The H2S donor NaHS inhibited spontaneous contractions of the longitudinal muscle and circular muscle strips in a dose-dependent manner, and the inhibitory effects were not blocked by tetrodotoxin. NaHS inhibited the peak ICa,L in colonic SMCs at a membrane potential of 0 mV. The current-voltage (I-V) relationship of L-type calcium channels was modified by NaHS, and the peak of the I-V curve was shifted to the right. NaHS (200 μΜ) evoked a significant rightward shift of the steady-state activation curve and inhibited the inactivation of L-type calcium channels. Furthermore, NaHS reversibly decreased the peak ICa,L in a dose-dependent manner. Likewise, BKCa channels were significantly inhibited by NaHS, and the addition of NaHS caused a time- and dose-dependent reduction in the BKCa current., Conclusion: The relaxant effect of H2S on colonic muscle strips may be associated with the direct inhibition of H2S on L-type calcium channels. H2S may be involved in the regulation of calcium homeostasis in colonic SMCs of rat colon.

Formaldehyde (FA) is a common environmental contaminant that has toxic effects on the central nervous system (CNS). Our previous data demonstrated that hydrogen sulfide (H2S), the third endogenous gaseous mediator, has protective effects against FA-induced neurotoxicity. As is known to all, Brain-derived neurotropic factor (BDNF), a member of the neurotrophin gene family, mediates its neuroprotective properties via various intracellular signaling pathways triggered by activating the tyrosine kinase receptor B (TrkB). Intriguingly, our previous data have illustrated the upregulatory role of H2S on BDNF protein expression in the hippocampus of rats. Therefore, in this study, we hypothesized that H2S provides neuroprotection against FA toxicity by regulating BDNF-TrkB pathway. In the present study, we found that NaHS, a donor of H2S, upregulated the level of BDNF protein in PC12 cells, and significantly rescued FA-induced downregulation of BDNF levels. Furthermore, we found that pretreatment of PC12 cells with K252a, an inhibitor of the BDNF receptor TrkB, markedly reversed the inhibition of NaHS on FA-induced cytotoxicity and ablated the protective effects of NaHS on FA-induced oxidative stress, including the accumulation of intracellular reactive oxygen species (ROS), 4-hydroxy-2-trans-nonenal (4-HNE), and malondialdehyde (MDA). We also showed that K252a abolished the inhibition of NaHS on FA-induced apoptosis, as well as the activation of caspase-3 in PC12 cells. In addition, K252a reversed the protection of H2S against FA-induced downregulation of Bcl-2 protein expression and upregulation of Bax protein expression in PC12 cells. These data indicate that the BDNF-TrkB pathway mediates the neuroprotection of H2S against FA-induced cytotoxicity, oxidative stress and apoptosis in PC12 cells. These findings provide a novel mechanism underlying the protection of H2S against FA-induced neurotoxicity.

Porcine oocytes that have matured in in vitro conditions undergo the process of aging during prolonged cultivation, which is manifested by spontaneous parthenogenetic activation, lysis or fragmentation of aged oocytes. This study focused on the role of hydrogen sulfide (H2S) in the process of porcine oocyte aging. H2S is a gaseous signaling molecule and is produced endogenously by the enzymes cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST). We demonstrated that H2S-producing enzymes are active in porcine oocytes and that a statistically significant decline in endogenous H2S production occurs during the first day of aging. Inhibition of these enzymes accelerates signs of aging in oocytes and significantly increases the ratio of fragmented oocytes. The presence of exogenous H2S from a donor (Na2S.9H2O) significantly suppressed the manifestations of aging, reversed the effects of inhibitors and resulted in the complete suppression of oocyte fragmentation. Cultivation of aging oocytes in the presence of H2S donor positively affected their subsequent embryonic development following parthenogenetic activation. Although no unambiguous effects of exogenous H2S on MPF and MAPK activities were detected and the intracellular mechanism underlying H2S activity remains unclear, our study clearly demonstrates the role of H2S in the regulation of porcine oocyte aging.

Hydrogen sulfide (H2S) can protect the liver against ischemia-reperfusion (I/R) injury. However, it is unknown whether H2S plays a role in the protection of hepatic I/R injury in both young and old patients. This study compared the protective effects of H2S in a rat model (young and old animals) of I/R injury and the mechanism underlying its effects. Young and old rats were assessed following an injection of NaHS. NaHS alone reduced hepatic I/R injury in the young rats by activating the nuclear erythroid-related factor 2 (Nrf2) signaling pathway, but it had little effect on the old rats. NaHS pretreatment decreased miR-34a expression in the hepatocytes of the young rats with hepatic I/R. Overexpression of miR-34a decreased Nrf-2 and its downstream target expression, impairing the hepatoprotective effect of H2S on the young rats. More importantly, downregulation of miR-34a expression increased Nrf-2 and the expression of its downstream targets, enhancing the effect of H2S on hepatic I/R injury in the old rats. This study reveals the different effects of H2S on hepatic I/R injury in young and old rats and sheds light on the involvement of H2S in miR-34a modulation of the Nrf-2 pathway.

Hydrogen sulfide (H2S), as a potential gaseous messenger molecule, has been suggested to play important roles in a wide range of physiological processes in plants. The aim of present study was to investigate which set of proteins is involved in H2S-regulated metabolism or signaling pathways. Spinacia oleracea seedlings were treated with 100 µM NaHS, a donor of H2S. Changes in protein expression profiles were analyzed by 2-D gel electrophoresis coupled with MALDI-TOF MS. Over 1000 protein spots were reproducibly resolved, of which the abundance of 92 spots was changed by at least 2-fold (sixty-five were up-regulated, whereas 27 were down-regulated). These proteins were functionally divided into 9 groups, including energy production and photosynthesis, cell rescue, development and cell defense, substance metabolism, protein synthesis and folding, cellular signal transduction. Further, we found that these proteins were mainly localized in cell wall, plasma membrane, chloroplast, mitochondria, nucleus, peroxisome and cytosol. Our results demonstrate that H2S is involved in various cellular and physiological activities and has a distinct influence on photosynthesis, cell defense and cellular signal transduction in S. oleracea leaves. These findings provide new insights into proteomic responses in plants under physiological levels of H2S.

Hydrogen sulfide (H2S), a gasotransmitter endogenously found in the central nervous system, has recently been suggested to act as a signalling molecule in the brain having beneficial effects on cardiovascular function. This study was thus undertaken to investigate the effect of NaHS (an H2S donor) in the subfornical organ (SFO), a central nervous system site important to blood pressure regulation. We used male Sprague-Dawley rats for both in vivo and in vitro experiments. We first used RT-PCR to confirm our previous microarray analyses showing that mRNAs for the enzymes required to produce H2S are expressed in the SFO. We then used microinjection techniques to investigate the physiological effects of NaHS in SFO, and found that NaHS microinjection (5 nmol) significantly increased blood pressure (mean AUC = 853.5±105.7 mmHg*s, n = 5). Further, we used patch-clamp electrophysiology and found that 97.8% (88 of 90) of neurons depolarized in response to NaHS. This response was found to be concentration dependent with an EC50 of 35.6 µM. Coupled with the depolarized membrane potential, we observed an overall increase in neuronal excitability using an analysis of rheobase and action potential firing patterns. This study has provided the first evidence of NaHS and thus H2S actions and their cellular correlates in SFO, implicating this brain area as a site where H2S may act to control blood pressure.

In this research, the antifungal role of hydrogen sulfide (H2S) on the postharvest pathogens Aspergillus niger and Penicillium italicum growing on fruits and under culture conditions on defined media was investigated. Our results show that H2S, released by sodium hydrosulfide (NaHS) effectively reduced the postharvest decay of fruits induced by A. niger and P. italicum. Furthermore, H2S inhibited spore germination, germ tube elongation, mycelial growth, and produced abnormal mycelial contractions when the fungi were grown on defined media in Petri plates. Further studies showed that H2S could cause an increase in intracellular reactive oxygen species (ROS) in A. niger. In accordance with this observation we show that enzyme activities and the expression of superoxide dismutase (SOD) and catalase (CAT) genes in A. niger treated with H2S were lower than those in control. Moreover, H2S also significantly inhibited the growth of Saccharomyces cerevisiae, Rhizopus oryzae, the human pathogen Candida albicans, and several food-borne bacteria. We also found that short time exposure of H2S showed a microbicidal role rather than just inhibiting the growth of microbes. Taken together, this study suggests the potential value of H2S in reducing postharvest loss and food spoilage caused by microbe propagation.

Recently, we have shown that inhalation of hydrogen sulfide (H2S) protects against ventilator-induced lung injury (VILI). In the present study, we aimed to determine the underlying molecular mechanisms of H2S-dependent lung protection by analyzing gene expression profiles in mice. C57BL/6 mice were subjected to spontaneous breathing or mechanical ventilation in the absence or presence of H2S (80 parts per million). Gene expression profiles were determined by microarray, sqRT-PCR and Western Blot analyses. The association of Atf3 in protection against VILI was confirmed with a Vivo-Morpholino knockout model. Mechanical ventilation caused a significant lung inflammation and damage that was prevented in the presence of H2S. Mechanical ventilation favoured the expression of genes involved in inflammation, leukocyte activation and chemotaxis. In contrast, ventilation with H2S activated genes involved in extracellular matrix remodelling, angiogenesis, inhibition of apoptosis, and inflammation. Amongst others, H2S administration induced Atf3, an anti-inflammatory and anti-apoptotic regulator. Morpholino mediated reduction of Atf3 resulted in elevated lung injury despite the presence of H2S. In conclusion, lung protection by H2S during mechanical ventilation is associated with down-regulation of genes related to oxidative stress and inflammation and up-regulation of anti-apoptotic and anti-inflammatory genes. Here we show that Atf3 is clearly involved in H2S mediated protection.

Hydrogen sulfide (H2S) has been revealed to be a signal molecule with second messenger action in the somatic cells of many tissues, including the reproductive tract. The aim of this study was to address how exogenous H2S acts on the meiotic maturation of porcine oocytes, including key maturation factors such as MPF and MAPK, and cumulus expansion intensity of cumulus-oocyte complexes. We observed that the H2S donor, Na2S, accelerated oocyte in vitro maturation in a dose-dependent manner, following an increase of MPF activity around germinal vesicle breakdown. Concurrently, the H2S donor affected cumulus expansion, monitored by hyaluronic acid production. Our results suggest that the H2S donor influences oocyte maturation and thus also participates in the regulation of cumulus expansion. The exogenous H2S donor apparently affects key signal pathways of oocyte maturation and cumulus expansion, resulting in faster oocyte maturation with little need of cumulus expansion.

Hydrogen sulfide (H2S) is well known as a highly toxic environmental chemical threat. Prolonged exposure to H2S can lead to the formation of pulmonary edema. However, the mechanisms of how H2S facilitates edema formation are poorly understood. Since edema formation can be enhanced by an impaired clearance of electrolytes and, consequently, fluid across the alveolar epithelium, it was questioned whether H2S may interfere with transepithelial electrolyte absorption. Electrolyte absorption was electrophysiologically measured across native distal lung preparations (Xenopus laevis) in Ussing chambers. The exposure of lung epithelia to H2S decreased net transepithelial electrolyte absorption. This was due to an impairment of amiloride-sensitive sodium transport. H2S inhibited the activity of the Na+/K+-ATPase as well as lidocaine-sensitive potassium channels located in the basolateral membrane of the epithelium. Inhibition of these transport molecules diminishes the electrochemical gradient which is necessary for transepithelial sodium absorption. Since sodium absorption osmotically facilitates alveolar fluid clearance, interference of H2S with the epithelial transport machinery provides a mechanism which enhances edema formation in H2S-exposed lungs.

Background: Bronchopulmonary dysplasia (BPD), the chronic lung disease of prematurity, remains a major health problem. BPD is characterized by impaired alveolar development and complicated by pulmonary hypertension (PHT). Currently there is no specific treatment for BPD. Hydrogen sulfide (H2S), carbon monoxide and nitric oxide (NO), belong to a class of endogenously synthesized gaseous molecules referred to as gasotransmitters. While inhaled NO is already used for the treatment of neonatal PHT and currently tested for the prevention of BPD, H2S has until recently been regarded exclusively as a toxic gas. Recent evidence suggests that endogenous H2S exerts beneficial biological effects, including cytoprotection and vasodilatation. We hypothesized that H2S preserves normal alveolar development and prevents PHT in experimental BPD., Methods: We took advantage of a recently described slow-releasing H2S donor, GYY4137 (morpholin-4-ium-4-methoxyphenyl(morpholino) phosphinodithioate) to study its lung protective potential in vitro and in vivo., Results: In vitro, GYY4137 promoted capillary-like network formation, viability and reduced reactive oxygen species in hyperoxia-exposed human pulmonary artery endothelial cells. GYY4137 also protected mitochondrial function in alveolar epithelial cells. In vivo, GYY4137 preserved and restored normal alveolar growth in rat pups exposed from birth for 2 weeks to hyperoxia. GYY4137 also attenuated PHT as determined by improved pulmonary arterial acceleration time on echo-Doppler, pulmonary artery remodeling and right ventricular hypertrophy. GYY4137 also prevented pulmonary artery smooth muscle cell proliferation., Conclusions: H2S protects from impaired alveolar growth and PHT in experimental O2-induced lung injury. H2S warrants further investigation as a new therapeutic target for alveolar damage and PHT.

Background: Formaldehyde (FA), a well-known environmental pollutant, has been classified as a neurotoxic molecule. Our recent data demonstrate that hydrogen sulfide (H2S), the third gaseous transmitter, has a protective effect on the neurotoxicity of FA. However, the exact mechanisms underlying this protection remain largely unknown. Endoplasmic reticulum (ER) stress has been implicated in the neurotoxicity of FA. Silent mating type information regulator 2 homolog 1 (SIRT-1), a histone deacetylases, has various biological activities, including the extension of lifespan, the modulation of ER stress, and the neuroprotective action., Objective: We hypothesize that the protection of H2S against FA-induced neurotoxicity involves in inhibiting ER stress by upregulation of SIRT-1. The present study attempted to investigate the protective effect of H2S on FA-induced ER stress in PC12 cells and the contribution of SIRT-1 to the protection of H2S against FA-induced injuries, including ER stress, cytotoxicity and apoptosis., Principal Findings: We found that exogenous application of sodium hydrosulfide (NaHS; an H2S donor) significantly attenuated FA-induced ER stress responses, including the upregulated levels of glucose-regulated protein 78, C/EBP homologous protein, and cleaved caspase-12 expression. We showed that NaHS upregulates the expression of SIRT-1 in PC12 cells. Moreover, the protective effects of H2S on FA-elicited ER stress, cytotoxicity and apoptosis were reversed by Sirtinol, a specific inhibitor of SIRT-1., Conclusion/significance: These data indicate that H2S exerts its protection against the neurotoxicity of FA through overcoming ER stress via upregulation of SIRT-1. Our findings provide novel insights into the protective mechanisms of H2S against FA-induced neurotoxicity.

Hydrogen sulfide (H2S) is an important gasotransmitter in mammals. Despite physiological changes induced by exogenous H2S donor NaHS to plants, whether and how H2S works as a true cellular signal in plants need to be examined. A self-developed specific fluorescent probe (WSP-1) was applied to track endogenous H2S in tomato (Solanum lycopersicum) roots in site. Bioimaging combined with pharmacological and biochemical approaches were used to investigate the cross-talk among H2S, nitric oxide (NO), and Ca(2+) in regulating lateral root formation. Endogenous H2S accumulation was clearly associated with primordium initiation and lateral root emergence. NO donor SNP stimulated the generation of endogenous H2S and the expression of the gene coding for the enzyme responsible for endogenous H2S synthesis. Scavenging H2S or inhibiting H2S synthesis partially blocked SNP-induced lateral root formation and the expression of lateral root-related genes. The stimulatory effect of SNP on Ca(2+) accumulation and CaM1 (calmodulin 1) expression could be abolished by inhibiting H2S synthesis. Ca(2+) chelator or Ca(2+) channel blocker attenuated NaHS-induced lateral root formation. Our study confirmed the role of H2S as a cellular signal in plants being a mediator between NO and Ca(2+) in regulating lateral root formation.

Hydrogen sulfide (H2S), a novel gaseous mediator, has been recognized as an important neuromodulator and neuroprotective agent in the central nervous system. The present study was undertaken to study the effects of exogenous H2S on traumatic brain injury (TBI) and the underlying mechanisms. The effects of exogenous H2S on TBI were examined by using measurement of brain edema, behavior assessment, propidium iodide (PI) staining, and Western blotting, respectively. Compared to TBI groups, H2S pretreatment had reduced brain edema, improved motor performance and ameliorated performance in Morris water maze test after TBI. Immunoblotting results showed that H2S pretreatment reversed TBI-induced cleavage of caspase-3 and decline of Bcl-2, suppressed LC3-II, Beclin-1 and Vps34 activation and maintained p62 level in injured cortex and hippocampus post TBI. The results suggest a protective effect and therapeutic potential of H2S in the treatment of brain injury and the protective effect against TBI may be associated with regulating apoptosis and autophagy.

Exogenous hydrogen sulfide (H2S) administration and endogenous H2S metabolism were explored in the nematode C. elegans. Chronic treatment with a slow-releasing H2S donor, GYY4137, extended median survival by 17-23% and increased tolerance towards oxidative and endoplasmic reticulum (ER) stress. Also, cysl-2, a sulfhydrylase/cysteine synthase in C. elegans, was transcriptionally upregulated by GYY4137 treatment and the deletion of cysl-2 resulted in a significant reduction in lifespan which was partially recovered by the supplementation of GYY4137. Likewise, a mammalian cell culture system, GYY4137 was able to protect bovine aortic endothelial cells (BAECs) from oxidative stress and (H2O2)-induced cell death. Taken together, this provides further support that H2S exerts a protective function which is consistent with the longevity dividend theory. Overall, this study underlines the therapeutic potential of a slow-releasing H2S donor as regulators of the aging and cellular stress pathways.

Hydrogen sulfide (H2S) is a gasotransmitter and plays an important role in many physiological processes in mammals. Studies of its functions in plants are attracting ever growing interest, for example, its ability to enhance drought resistance in Arabidopsis. A general role of microRNAs (miRNAs) in plant adaptive responses to drought stress has thereby increased our interest to delve into the possible interplay between H2S and miRNAs. Our results showed that treating wild type (WT) Arabidopsis seedlings with polyethylene glycol 8000 (PEG8000) to simulate drought stress caused an increase in production rate of endogenous H2S; and a significant transcriptional reformation of relevant miRNAs, which were also triggered by exogenous H2S in WT. When lcd mutants (with lower H2S production rate than WT) were treated with PEG8000, they showed lower levels of miRNA expression changes than WT. In addition, we detected significant changes in target gene expression of those miRNAs and the corresponding phenotypes in lcd, including less roots, retardation of leaf growth and development and greater superoxide dismutase (SOD) activity under drought stress. We thereby conclude that H2S can improve drought resistance through regulating drought associated miRNAs in Arabidopsis.

Hydrogen sulfide (H2S) is the third most common endogenously produced gaseous signaling molecule, but its impact on hepatic ischemia/reperfusion (I/R) injury, especially on mitochondrial function, remains unclear. In this study, rats were randomized into Sham, I/R, ischemia preconditioning (IPC) or sodium hydrosulfide (NaHS, an H2S donor) preconditioning groups. To establish a model of segmental (70%) warm hepatic ischemia, the hepatic artery, left portal vein and median liver lobes were occluded for 60 min and then unclamped to allow reperfusion. Preconditioning with 12.5, 25 or 50 μmol/kg NaHS prior to the I/R insult significantly increased serum H2S levels, and, similar to IPC, NaHS preconditioning decreased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the plasma and prevented hepatocytes from undergoing I/R-induced necrosis. Moreover, a sub-toxic dose of NaHS (25 μmol/kg) did not disrupt the systemic hemodynamics but dramatically inhibited mitochondrial permeability transition pore (MPTP) opening and thus prevented mitochondrial-related cell death and apoptosis. Mechanistic studies revealed that NaHS preconditioning markedly increased the expression of phosphorylated protein kinase B (p-Akt), phosphorylated glycogen synthase kinase-3 beta (p-GSK-3β) and B-cell lymphoma-2 (Bcl-2) and decreased the release of mitochondrial cytochrome c and cleaved caspase-3/9 levels. Therefore, NaHS administration prior to hepatic I/R ameliorates mitochondrial and hepatocellular damage through the inhibition of MPTP opening and the activation of Akt-GSK-3β signaling. Furthermore, this study provides experimental evidence for the clinical use of H2S to reduce liver damage after perioperative I/R injury.

Decrease in endogenous hydrogen sulfide (H2S) was reported to participate in the pathogenesis of diabetic nephropathy (DN). This study is aimed at exploring the relationship between the abnormalities in H2S metabolism, hyperglycemia-induced oxidative stress and the activation of intrarenal renin-angiotensin system (RAS). Cultured renal mesangial cells (MCs) and streptozotocin (STZ) induced diabetic rats were used for the studies. The expressions of angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin II (Ang II) type I receptor (AT1), transforming growth factor-β1 (TGF-β1) and collagen IV were measured by real time PCR and Western blot. Reactive oxygen species (ROS) production was assessed by fluorescent probe assays. Cell proliferation was analyzed by 5'-bromo-2'-deoxyuridine incorporation assay. Ang II concentration was measured by an enzyme immunoassay. AGT, ACE and AT1 receptor mRNA levels and Ang II concentration were increased in high glucose (HG) -treated MCs, the cell proliferation rate and the production of TGF-β1 and of collagen IV productions were also increased. The NADPH oxidase inhibitor diphenylenechloride iodonium (DPI) was able to reverse the HG-induced RAS activation and the changes in cell proliferation and collagen synthesis. Supplementation of H2S attenuated HG-induced elevations in ROS and RAS activation. Blockade on H2S biosynthesis from cystathione-γ-lyase (CSE) by DL-propargylglycine (PPG) resulted in effects similar to that of HG treatment. In STZ-induced diabetic rats, the changes in RAS were also reversed by H2S supplementation without affecting blood glucose concentration. These data suggested that the decrease in H2S under hyperglycemic condition leads to an imbalance between oxidative and reductive species. The increased oxidative species results in intrarenal RAS activation, which, in turn, contributes to the pathogenesis of renal dysfunction.

Objective: Heart failure (HF) is one of the most serious diseases worldwide. S-propargyl-cysteine (SPRC), a novel modulator of endogenous hydrogen sulfide, is proved to be able to protect against acute myocardial ischemia. In order to produce more stable and sustainable hydrogen sulfide, we used controlled release formulation of SPRC (CR-SPRC) to elucidate possible cardioprotective effects on HF rats and investigate involved mechanisms on apoptosis and oxidation., Methods: Left coronary artery was occluded to induce HF model of rat. The survival rats were randomly divided into 7 groups after 24 hours and treated with drugs for 6 weeks. Echocardiographic indexes were recorded to determine cardiac function. TTC staining was performed to determine infarct size. Plasmatic level of hydrogen sulfide was detected by modified sulfide electrode. Activity of enzyme and expression of protein were determined by colorimetry and Western blot, respectively., Results: The cardioprotective effects of CR-SPRC on HF rats were confirmed by significant reduction of infarct size and improvement of cardiac function, with better effects compared to normal SPRC. CR-SPRC modulated antioxidant defenses by preserving levels of GSH, CAT and SOD and reducing CK leakage. In addition, CR-SPRC elevated ratio of Bcl-2/Bax and inhibited activity of caspases to protect against myocardial apoptosis. The cardioprotective effects of CR-SPRC were mediated by hydrogen sulfide., Conclusions: All experiment data indicated cardioprotective effects of CR-SPRC on HF rats. More importantly, CR-SPRC exerted better effects than normal SPRC in all respects, providing a new perspective on hydrogen sulfide-mediated drug therapy.

Sodium reabsorption through the epithelial sodium channel (ENaC) at the distal segment of the kidney plays an important role in salt-sensitive hypertension. We reported previously that hydrogen peroxide (H2O2) stimulates ENaC in A6 distal nephron cells via elevation of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) in the apical membrane. Here we report that H2S can antagonize H2O2-induced activation of ENaC in A6 cells. Our cell-attached patch-clamp data show that ENaC open probability (PO ) was significantly increased by exogenous H2O2, which is consistent with our previous finding. The aberrant activation of ENaC induced by exogenous H2O2 was completely abolished by H2S (0.1 mM NaHS). Pre-treatment of A6 cells with H2S slightly decreased ENaC P(O); however, in these cells H2O2 failed to elevate ENaC PO . Confocal microscopy data show that application of exogenous H2O2 to A6 cells significantly increased intracellular reactive oxygen species (ROS) level and induced accumulation of PI(3,4,5)P3 in the apical compartment of the cell membrane. These effects of exogenous H2O2 on intracellular ROS levels and on apical PI(3,4,5)P3 levels were almost completely abolished by treatment of A6 cells with H2S. In addition, H2S significantly inhibited H2O2-induced oxidative inactivation of the tumor suppressor phosphatase and tensin homolog (PTEN) which is a negative regulator of PI(3,4,5)P3. Moreover, BPV(pic), a specific inhibitor of PTEN, elevated PI(3,4,5)P3 and ENaC activity in a manner similar to that of H2O2 in A6 cells. Our data show, for the first time, that H2S prevents H2O2-induced activation of ENaC through a PTEN-PI(3,4,5)P3 dependent pathway.

This study presents a novel way of enhancing plant growth through the use of a non-petroleum based product. We report here that exposing either roots or seeds of multicellular plants to extremely low concentrations of dissolved hydrogen sulfide at any stage of life causes statistically significant increases in biomass including higher fruit yield. Individual cells in treated plants were smaller (~13%) than those of controls. Germination success and seedling size increased in, bean, corn, wheat, and pea seeds while time to germination decreases. These findings indicated an important role of H2S as a signaling molecule that can increase the growth rate of all species yet tested. The increased crop yields reported here has the potential to effect the world's agricultural output.

Background: Hydrogen sulfide (H₂S) has been shown to have cytoprotective effects in models of hypertension, ischemia/reperfusion and Alzheimer's disease. However, little is known about its effects or mechanisms of action in atherosclerosis. Therefore, in the current study we evaluated the pharmacological effects of H₂S on antioxidant defenses and mitochondria protection against hydrogen peroxide (H₂O₂) induced endothelial cells damage., Methodology and Principal Findings: H₂S, at non-cytotoxic levels, exerts a concentration dependent protective effect in human umbilical vein endothelial cells (HUVECs) exposed to H₂O₂. Analysis of ATP synthesis, mitochondrial membrane potential (ΔΨm) and cytochrome c release from mitochondria indicated that mitochondrial function was preserved by pretreatment with H₂S. In contrast, in H₂O₂ exposed endothelial cells mitochondria appeared swollen or ruptured. In additional experiments, H₂S was also found to preserve the activities and protein expressions levels of the antioxidants enzymes, superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase in H₂O₂ exposed cells. ROS and lipid peroxidation, as assessed by measuring H₂DCFDA, dihydroethidium (DHE), diphenyl-l-pyrenylphosphine (DPPP) and malonaldehyde (MDA) levels, were also inhibited by H₂S treatment. Interestingly, in the current model, D, L-propargylglycine (PAG), a selective inhibitor of cystathionine γ-lyase (CSE), abolished the protective effects of H₂S donors., Innovation: This study is the first to show that H₂S can inhibit H₂O₂ mediated mitochondrial dysfunction in human endothelial cells by preserving antioxidant defences., Significance: H₂S may protect against atherosclerosis by preventing H₂O₂ induced injury to endothelial cells. These effects appear to be mediated via the preservation of mitochondrial function and by reducing the deleterious effects of oxidative stress.

Objective: To investigate the potential role of hydrogen sulphide (H(2)S) and ATP-sensitive potassium (K(ATP)) channels in chronic stress-induced colonic hypermotility., Methods: Male Wistar rats were submitted daily to 1 h of water avoidance stress (WAS) or sham WAS (SWAS) for 10 consecutive days. Organ bath recordings, H(2)S production, immunohistochemistry and western blotting were performed on rat colonic samples to investigate the role of endogenous H(2)S in repeated WAS-induced hypermotility. Organ bath recordings and western blotting were used to detect the role of K(ATP) channels in repeated WAS., Results: Repeated WAS increased the number of fecal pellets per hour and the area under the curve of the spontaneous contractions of colonic strips, and decreased the endogenous production of H(2)S and the expression of H(2)S-producing enzymes in the colon devoid of mucosa and submucosa. Inhibitors of H(2)S-producing enzymes increased the contractile activity of colonic strips in the SWAS rats. NaHS concentration-dependently inhibited the spontaneous contractions of the strips and the NaHS IC(50) for the WAS rats was significantly lower than that for the SWAS rats. The inhibitory effect of NaHS was significantly reduced by glybenclamide. Repeated WAS treatment resulted in up-regulation of Kir6.1 and SUR2B of K(ATP) channels in the colon devoid of mucosa and submucosa., Conclusion: The colonic hypermotility induced by repeated WAS may be associated with the decreased production of endogenous H(2)S. The increased expression of the subunits of K(ATP) channels in colonic smooth muscle cells may be a defensive response to repeated WAS. H(2)S donor may have potential clinical utility in treating chronic stress-induced colonic hypermotility.

Introduction: Cerebral malaria (CM) is a potentially fatal cerebrovascular disease of complex pathogenesis caused by Plasmodium falciparum. Hydrogen sulfide (HS) is a physiological gas, similar to nitric oxide and carbon monoxide, involved in cellular metabolism, vascular tension, inflammation, and cell death. HS treatment has shown promising results as a therapy for cardio- and neuro- pathology. This study investigates the effects of fast (NaHS) and slow (GYY4137) HS-releasing drugs on the growth and metabolism of P. falciparum and the development of P. berghei ANKA CM. Moreover, we investigate the role of free plasma thiols and cell surface thiols in the pathogenesis of CM., Methods: P. falciparum was cultured in vitro with varying doses of HS releasing drugs compared with artesunate. Growth and metabolism were quantified. C57Bl/6 mice were infected with P. berghei ANKA and were treated with varying doses and regimes of HS-releasing drugs. Free plasma thiols and cell surface thiols were quantified in CM mice and age-matched healthy controls., Results: HS-releasing drugs significantly and dose-dependently inhibited P. falciparum growth and metabolism. Treatment of CM did not affect P. berghei growth, or development of CM. Interestingly, CM was associated with lower free plasma thiols, reduced leukocyte+erythrocyte cell surface thiols (infection day 3), and markedly (5-fold) increased platelet cell surface thiols (infection day 7)., Conclusions: HS inhibits P. falciparum growth and metabolism in vitro. Reduction in free plasma thiols, cell surface thiols and a marked increase in platelet cell surface thiols are associated with development of CM. HS drugs were not effective in vivo against murine CM.

Hydrogen sulfide (H(2)S) is a gaseous bacterial metabolite that reaches high levels in the large intestine. In the present study, the effect of H(2)S on the proliferation of normal and cancerous colon epithelial cells was investigated. An immortalized colon epithelial cell line (YAMC) and a panel of colon cancer cell lines (HT-29, SW1116, HCT116) were exposed to H(2)S at concentrations similar to those found in the human colon. H(2)S inhibited normal and cancerous colon epithelial cell proliferation as measured by MTT assay. The anti-mitogenic effect of H(2)S was accompanied by G(1)-phase cell cycle arrest and the induction of the cyclin-dependent kinase inhibitor p21(Cip). Moreover, exposure to H(2)S led to features characteristic of autophagy, including increased formation of LC3B(+) autophagic vacuoles and acidic vesicular organelles as determined by immunofluorescence and acridine orange staining, respectively. Abolition of autophagy by RNA interference targeting Vps34 or Atg7 enhanced the anti-proliferative effect of H(2)S. Further mechanistic investigation revealed that H(2)S stimulated the phosphorylation of AMP-activated protein kinase (AMPK) and inhibited the phosphorylation of mammalian target of rapamycin (mTOR) and S6 kinase. Inhibition of AMPK significantly reversed H(2)S-induced autophagy and inhibition of cell proliferation. Collectively, we demonstrate that H(2)S inhibits colon epithelial cell proliferation and induces protective autophagy via the AMPK pathway.

Hydrogen sulfide (H(2)S), which is produced endogenously from L-cysteine, is an irritant with pro-nociceptive actions. We have used measurements of intracellular calcium concentration, electrophysiology and behavioral measurements to show that the somatic pronociceptive actions of H(2)S require TRPA1. A H(2)S donor, NaHS, activated TRPA1 expressed in CHO cells and stimulated DRG neurons isolated from Trpa1(+/+) but not Trpa1(-/-) mice. TRPA1 activation by NaHS was pH dependent with increased activity at acidic pH. The midpoint of the relationship between NaHS EC(50) values and external pH was pH 7.21, close to the expected dissociation constant for H(2)S (pK(a) 7.04). NaHS evoked single channel currents in inside-out and cell-attached membrane patches consistent with an intracellular site of action. In behavioral experiments, intraplantar administration of NaHS and L-cysteine evoked mechanical and cold hypersensitivities in Trpa1(+/+) but not in Trpa1(-/-) mice. The sensitizing effects of L-cysteine in wild-type mice were inhibited by a cystathionine β-synthase inhibitor, D,L-propargylglycine (PAG), which inhibits H(2)S formation. Mechanical hypersensitivity evoked by intraplantar injections of LPS was prevented by PAG and the TRPA1 antagonist AP-18 and was absent in Trpa1(-/-) mice, indicating that H(2)S mediated stimulation of TRPA1 is necessary for the local pronociceptive effects of LPS. The pro-nociceptive effects of intraplantar NaHS were retained in Trpv1(-/-) mice ruling out TRPV1 as a molecular target. In behavioral studies, NaHS mediated sensitization was also inhibited by a T-type calcium channel inhibitor, mibefradil. In contrast to the effects of NaHS on somatic sensitivity, intracolonic NaHS administration evoked similar nociceptive effects in Trpa1(+/+) and Trpa1(-/-) mice, suggesting that the visceral pro-nociceptive effects of H(2)S are independent of TRPA1. In electrophysiological studies, the depolarizing actions of H(2)S on isolated DRG neurons were inhibited by AP-18, but not by mibefradil indicating that the primary excitatory effect of H(2)S on DRG neurons is TRPA1 mediated depolarization.

Increasing evidence about the central nervous representation of pain in the brain suggests that the operculo-insular cortex is a crucial part of the pain matrix. The pain-specificity of a brain region may be tested by administering nociceptive stimuli while controlling for unspecific activations by administering non-nociceptive stimuli. We applied this paradigm to nasal chemosensation, delivering trigeminal or olfactory stimuli, to verify the pain-specificity of the operculo-insular cortex. In detail, brain activations due to intranasal stimulation induced by non-nociceptive olfactory stimuli of hydrogen sulfide (5 ppm) or vanillin (0.8 ppm) were used to mask brain activations due to somatosensory, clearly nociceptive trigeminal stimulations with gaseous carbon dioxide (75% v/v). Functional magnetic resonance (fMRI) images were recorded from 12 healthy volunteers in a 3T head scanner during stimulus administration using an event-related design. We found that significantly more activations following nociceptive than non-nociceptive stimuli were localized bilaterally in two restricted clusters in the brain containing the primary and secondary somatosensory areas and the insular cortices consistent with the operculo-insular cortex. However, these activations completely disappeared when eliminating activations associated with the administration of olfactory stimuli, which were small but measurable. While the present experiments verify that the operculo-insular cortex plays a role in the processing of nociceptive input, they also show that it is not a pain-exclusive brain region and allow, in the experimental context, for the interpretation that the operculo-insular cortex splay a major role in the detection of and responding to salient events, whether or not these events are nociceptive or painful.

Hydrogen sulfide (H(2)S), a novel gaseous messenger, is synthesized endogenously from L-cysteine by two pyridoxal-5'-phosphate-dependent enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). S-propargyl-cysteine (SPRC) is a slow H(2)S releasing drug that provides cysteine, a substrate of CSE. The present study was aimed to investigate the effects of SPRC in an in vivo model of acute pancreatitis (AP) in mice. AP was induced in mice by hourly caerulein injections (50 µg/kg) for 10 hours. Mice were treated with SPRC (10 mg/kg) or vehicle (distilled water). SPRC was administered either 12 h before or 3 h before the induction of pancreatitis. Mice were sacrificed 1 h after the last caerulein injection. Blood, pancreas and lung tissues were collected and processed to measure the plasma amylase, plasma H(2)S, myeloperoxidase (MPO) activities and cytokine levels in pancreas and lung. The results revealed that significant reduction of inflammation, both in pancreas and lung was associated with SPRC given 3 h prior to the induction of AP. Furthermore, the beneficial effects of SPRC were associated with reduction of pancreatic and pulmonary pro-inflammatory cytokines and increase of anti-inflammatory cytokine. SPRC administered 12 h before AP induction did not cause significant improvement in pancreatic and lung inflammation. Plasma H(2)S concentration showed significant difference in H(2)S levels between control, vehicle and SPRC (administered 3 h before AP) treatment groups. In conclusion, these data provide evidence for protective effects of SPRC in AP possibly by virtue of its slow release of endogenous H(2)S.

Better tocolytics are required to help prevent preterm labour. The gaseotransmitter Hydrogen sulphide (H(2)S) has been shown to reduce myometrial contractility and thus is of potential interest. However previous studies used NaHS, which is toxic and releases H(2)S as a non-physiological bolus and thus alternative H(2)S donors are sought. GYY4137 has been developed to slowly release H(2)S and hence better reflect endogenous physiological release. We have examined its effects on spontaneous and oxytocin-stimulated contractility and compared them to NaHS, in human and rat myometrium, throughout gestation. The effects on contractility in response to GYY4137 (1 nM-1 mM) and NaHS (1 mM) were examined on myometrial strips from, biopsies of women undergoing elective caesarean section or hysterectomy, and from non-pregnant, 14, 18, 22 day (term) gestation or labouring rats. In pregnant rat and human myometrium dose-dependent and significant decreases in spontaneous contractions were seen with increasing concentrations of GYY4137, which also reduced underlying Ca transients. GYY4137 and NaHS significantly reduced oxytocin-stimulated and high-K depolarised contractions as well as spontaneous activity. Their inhibitory effects increased as gestation advanced, but were abruptly reversed in labour. Glibenclamide, an inhibitor of ATP-sensitive potassium (K(ATP)) channels, abolished the inhibitory effect of GYY4137. These data suggest (i) H(2)S contributes to uterine quiescence from mid-gestation until labor, (ii) that H(2)S affects L-type calcium channels and K(ATP) channels reducing Ca entry and thereby myometrial contractions, (iii) add to the evidence that H(2)S plays a physiological role in relaxing myometrium, and thus (iv) H(2)S is an attractive target for therapeutic manipulation of human myometrial contractility.

Background: Hydrogen sulfide (H(2)S) is a potent vasodilator. However, the complex mechanisms of vasoregulation by H(2)S are not fully understood. We tested the hypotheses that (1) H(2)S exerts vasodilatory effects by opening KCNQ-type voltage-dependent (K(v)) K(+) channels and (2) that H(2)S-producing cystathionine-γ-lyase (CSE) in perivascular adipose tissue plays a major role in this pathway., Methodology/principal Findings: Wire myography of rat and mouse aortas was used. NaHS and 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH) were used as H(2)S donors. KCNQ-type K(v) channels were blocked by XE991. 4-Propargylglycine (PPG) and ß-cyano-l-alanine (BCA), or 2-(aminooxy)-acetic acid (AOAA) were used as inhibitors of CSE or cystathionine-ß-synthase (CBS), respectively. NaHS and ADTOH produced strong vasorelaxation in rat and mouse aortas, which were abolished by KCNQ channel inhibition with XE991. Perivascular adipose tissue (PVAT) exerted an anticontractile effect in these arteries. CSE inhibition by PPG and BCA reduced this effect in aortas from rats but not from mice. CBS inhibition with AOAA did not inhibit the anticontractile effects of PVAT. XE991, however, almost completely suppressed the anticontractile effects of PVAT in both species. Exogenous l-cysteine, substrate for the endogenous production of H(2)S, induced vasorelaxation only at concentrations >5 mmol/l, an effect unchanged by CSE inhibition., Conclusions/signficance: Our results demonstrate potent vasorelaxant effects of H(2)S donors in large arteries of both rats and mice, in which XE991-sensitive KCNQ-type channel opening play a pivotal role. CSE-H(2)S seems to modulate the effect of adipocyte-derived relaxing factor in rat but not in mouse aorta. The present study provides novel insight into the interaction of CSE-H(2)S and perivascular adipose tissue. Furthermore, with additional technical advances, a future clinical approach targeting vascular H(2)S/KCNQ pathways to influence states of vascular dysfunction may be possible.

Hydrogen sulfide (H(2)S) can be endogenously generated from cystathionine gamma-lyase (CSE) in cardiovascular system, offering a cardiovascular protection. It is also known that the lower risk of cardiovascular diseases in female is partially attributed to the protective effect of estrogen. The current study explores the interaction of H(2)S and estrogen on smooth muscle cell (SMC) growth. In the present study, we found that the proliferation of cultured vascular SMCs isolated from wild-type mice (WT-SMCs) was inhibited, but that from CSE gene knockout mice (CSE-KO-SMCs) increased, by estrogen treatments. The expression of estrogen receptor α (ERα), but not ERβ, was significantly decreased in CSE-KO-SMCs compared with that in WT-SMCs. Exogenously applied H(2)S markedly increased ERα but not ERβ expression. In addition, the inhibition of ER activation and knockdown of ERα expression in WT-SMCs or the overexpression of ERα in CSE-KO-SMCs reversed the respective effects of estrogen on cell proliferation. The expression of cyclin D1 was reduced in WT-SMCs but increased in CSE-KO-SMCs after estrogen treatments, which was reversed by knockdown of ERα in WT-SMCs or overexpression of ERα in CSE-KO-SMCs, respectively. The overexpression of cyclin D1 in WT-SMCs or knockdown of cyclin D1 expression in CSE-KO-SMCs reversed the effects of estrogen on cell proliferation. These results suggest that H(2)S mediates estrogen-inhibited proliferation of SMCs via selective activation of ERα/cyclin D1 pathways.

Hydrogen sulfide (H(2)S) is now considered as the third gaseotransmitter, however, the signaling pathways that modulate the biomedical effect of H(2)S on endothelial cells are poorly defined. In the present study, we found in human endothelial cells that H(2)S increased cell migration rates and induced a marked reorganization of the actin cytoskeleton, which was prevented by depletion of Rac1. Pharmacologic inhibiting vascular endothelial growth factor receptor (VEGFR) and phosphoinositide 3-kinase (PI3K) both blunted the activation of Rac1 and the promotion of cell migration induced by H(2)S. Moreover, H(2)S-induced Rac1 activation was selectively dependent on the presence of the PI3K p110α isoform. Activated Rac1 by H(2)S thus in turn resulted in the phosphorylation of the F-actin polymerization modulator, cofilin. Additionally, inhibiting of extracellular signal-regulated kinase (ERK) decreased the augmented cell migration rate by H(2)S, but had no effect on Rac1 activation. These results indicate that Rac1 conveys the H(2)S signal to microfilaments inducing rearrangements of actin cytoskeleton that regulates cell migration. VEGFR-PI3K was found to be upstream pathway of Rac1, while cofilin acted as a downstream effector of Rac1. ERK was also shown to be involved in the action of H(2)S on endothelial cell migration, but independently of Rac1.

Hydrogen sulfide, as a novel gaseous mediator, has been suggested to play a key role in atherogenesis. However, the precise mechanisms by which H(2)S affects atherosclerosis remain unclear. Therefore, the present study aimed to investigate the potential role of H(2)S in atherosclerosis and the underlying mechanism with respect to chemokines (CCL2, CCL5 and CX3CL1) and chemokine receptors (CCR2, CCR5, and CX3CR1) in macrophages. Mouse macrophage cell line RAW 264.7 or mouse peritoneal macrophages were pre-incubated with saline or NaHS (50 µM, 100 µM, 200 µM), an H(2)S donor, and then stimulated with interferon-γ (IFN-γ) or lipopolysaccharide (LPS). It was found that NaHS dose-dependently inhibited IFN-γ or LPS-induced CX3CR1 and CX3CL1 expression, as well as CX3CR1-mediated chemotaxis in macrophages. Overexpression of cystathionine γ-lyase (CSE), an enzyme that catalyzes H(2)S biosynthesis resulted in a significant reduction in CX3CR1 and CX3CL1 expression as well as CX3CR1-mediated chemotaxis in stimulated macrophages. The inhibitory effect of H(2)S on CX3CR1 and CX3CL1 expression was mediated by modulation of proliferators-activated receptor-γ (PPAR-γ) and NF-κB pathway. Furthermore, male apoE(-/-) mice were fed a high-fat diet and then randomly given NaHS (1 mg/kg, i.p., daily) or DL-propargylglycine (PAG, 10 mg/kg, i.p., daily). NaHS significantly inhibited aortic CX3CR1 and CX3CL1 expression and impeded aortic plaque development. NaHS had a better anti-atherogenic benefit when it was applied at the early stage of atherosclerosis. However, inhibition of H(2)S formation by PAG increased aortic CX3CR1 and CX3CL1 expression and exacerbated the extent of atherosclerosis. In addition, H(2)S had minimal effect on the expression of CCL2, CCL5, CCR2 and CCR5 in vitro and in vivo. In conclusion, these data indicate that H(2)S hampers the progression of atherosclerosis in fat-fed apoE(-/-) mice and downregulates CX3CR1 and CX3CL1 expression on macrophages and in lesion plaques.

Aim: Hydrogen sulfide (H₂S) is a promising cardioprotective agent and a potential modulator of cardiac ion currents. Yet its cardiac effects on humans are poorly understood due to lack of functional cardiomyocytes. This study investigates electrophysiological responses of human pluripotent stem cells (hPSCs) derived cardiomyocytes towards H₂S., Methods and Results: Cardiomyocytes of ventricular, atrial and nodal subtypes differentiated from H9 embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) were electrophysiologically characterized. The effect of NaHS, a donor of H₂S, on action potential (AP), outward rectifier potassium currents (I(Ks) and I(Kr)), L-type Ca²⁺ currents (I(CaL)) and hyperpolarization-activated inward current (I(f)) were determined by patch-clamp electrophysiology and confocal calcium imaging. In a concentration-dependent manner, NaHS (100 to 300 µM) consistently altered the action potential properties including prolonging action potential duration (APD) and slowing down contracting rates of ventricular-and atrial-like cardiomyocytes derived from both hESCs and hiPSCs. Moreover, inhibitions of slow and rapid I(K) (I(Ks) and I(Kr)), I(CaL) and I(f) were found in NaHS treated cardiomyocytes and it could collectively contribute to the remodeling of AP properties., Conclusions: This is the first demonstration of effects of H₂S on cardiac electrophysiology of human ventricular-like, atrial-like and nodal-like cardiomyocytes. It reaffirmed the inhibitory effect of H₂S on I(CaL) and revealed additional novel inhibitory effects on I(f), I(Ks) and I(Kr) currents in human cardiomyocytes.

Hydrogen sulfide (H(2)S) is a novel gasotransmitter that inhibits L-type calcium currents (I (Ca, L)). However, the underlying molecular mechanisms are unclear. In particular, the targeting site in the L-type calcium channel where H(2)S functions remains unknown. The study was designed to investigate if the sulfhydryl group could be the possible targeting site in the L-type calcium channel in rat cardiomyocytes. Cardiac function was measured in isolated perfused rat hearts. The L-type calcium currents were recorded by using a whole cell voltage clamp technique on the isolated cardiomyocytes. The L-type calcium channel containing free sulfhydryl groups in H9C2 cells were measured by using Western blot. The results showed that sodium hydrosulfide (NaHS, an H(2)S donor) produced a negative inotropic effect on cardiac function, which could be partly inhibited by the oxidant sulfhydryl modifier diamide (DM). H(2)S donor inhibited the peak amplitude of I( Ca, L) in a concentration-dependent manner. However, dithiothreitol (DTT), a reducing sulfhydryl modifier markedly reversed the H(2)S donor-induced inhibition of I (Ca, L) in cardiomyocytes. In contrast, in the presence of DM, H(2)S donor could not alter cardiac function and L type calcium currents. After the isolated rat heart or the cardiomyocytes were treated with DTT, NaHS could markedly alter cardiac function and L-type calcium currents in cardiomyocytes. Furthermore, NaHS could decrease the functional free sulfhydryl group in the L-type Ca(2+) channel, which could be reversed by thiol reductant, either DTT or reduced glutathione. Therefore, our results suggest that H(2)S might inhibit L-type calcium currents depending on the sulfhydryl group in rat cardiomyocytes.

A growing body of evidence suggests that hydrogen sulfide (H₂S) is a signaling molecule in mammalian cells. In the cardiovascular system, H₂S enhances vasodilation and angiogenesis. H₂S-induced vasodilation is hypothesized to occur through ATP-sensitive potassium channels (K(ATP)); however, we recently demonstrated that it also increases cGMP levels in tissues. Herein, we studied the involvement of cGMP-dependent protein kinase-I in H₂S-induced vasorelaxation. The effect of H₂S on vessel tone was studied in phenylephrine-contracted aortic rings with or without endothelium. cGMP levels were determined in cultured cells or isolated vessel by enzyme immunoassay. Pretreatment of aortic rings with sildenafil attenuated NaHS-induced relaxation, confirming previous findings that H₂S is a phosphodiesterase inhibitor. In addition, vascular tissue levels of cGMP in cystathionine gamma lyase knockouts were lower than those in wild-type control mice. Treatment of aortic rings with NaHS, a fast releasing H₂S donor, enhanced phosphorylation of vasodilator-stimulated phosphoprotein in a time-dependent manner, suggesting that cGMP-dependent protein kinase (PKG) is activated after exposure to H₂S. Incubation of aortic rings with a PKG-I inhibitor (DT-2) attenuated NaHS-stimulated relaxation. Interestingly, vasodilatory responses to a slowly releasing H₂S donor (GYY 4137) were unaffected by DT-2, suggesting that this donor dilates mouse aorta through PKG-independent pathways. Dilatory responses to NaHS and L-cysteine (a substrate for H₂S production) were reduced in vessels of PKG-I knockout mice (PKG-I⁻/⁻). Moreover, glibenclamide inhibited NaHS-induced vasorelaxation in vessels from wild-type animals, but not PKG-I⁻/⁻, suggesting that there is a cross-talk between K(ATP) and PKG. Our results confirm the role of cGMP in the vascular responses to NaHS and demonstrate that genetic deletion of PKG-I attenuates NaHS and L-cysteine-stimulated vasodilation.

Background: Intracerebroventricular infusion of NaHS, a hydrogen sulfide (H(2)S) donor, increased mean arterial pressure (MAP). This study was designed to determine the roles of H(2)S in the paraventricular nucleus (PVN) in modulating sympathetic activity and cardiac sympathetic afferent reflex (CSAR) in chronic heart failure (CHF)., Methodology/principal Findings: CHF was induced by left descending coronary artery ligation in rats. Renal sympathetic nerve activity (RSNA) and MAP were recorded under anesthesia. CSAR was evaluated by the RSNA and MAP responses to epicardial application of capsaicin. PVN microinjection of low doses of a H(2)S donor, GYY4137 (0.01 and 0.1 nmol), had no significant effects on RSNA, MAP and CSAR. High doses of GYY4137 (1, 2 and 4 nmol) increased baseline RSNA, MAP and heart rate (HR), and enhanced CSAR. The effects were greater in CHF rats than sham-operated rats. A cystathionine-β-synthase (CBS) inhibitor, hydroxylamine (HA) in PVN had no significant effect on the RSNA, MAP and CSAR. CBS activity and H(2)S level in the PVN were decreased in CHF rats. No significant difference in CBS level in PVN was found between sham-operated rats and CHF rats. Stimulation of cardiac sympathetic afferents with capsaicin decreased CBS activity and H(2)S level in the PVN in both sham-operated rats and CHF rats., Conclusions: Exogenous H(2)S in PVN increases RSNA, MAP and HR, and enhances CSAR. The effects are greater in CHF rats than those in sham-operated rats. Endogenous H(2)S in PVN is not responsible for the sympathetic activation and enhanced CSAR in CHF rats.

Background: Hydrogen sulfide (H(2)S) is a newly identified member of the small family of gasotransmitters that are endogenous gaseous signaling molecules that have a fundamental role in human biology and disease. Although it is a relatively recent discovery and the mechanism of H(2)S activity is not completely understood, it is known to be involved in a number of cellular processes; H(2)S can affect ion channels, transcription factors and protein kinases in mammals., Methodology/principal Findings: In this paper, we have used fission yeast as a model organism to study the global gene expression profile in response to H(2)S by microarray. We initially measured the genome-wide transcriptional response of fission yeast to H(2)S. Through the functional classification of genes whose expression profile changed in response to H(2)S, we found that H(2)S mainly influences genes that encode putative or known stress proteins, membrane transporters, cell cycle/meiotic proteins, transcription factors and respiration protein in the mitochondrion. Our analysis showed that there was a significant overlap between the genes affected by H(2)S and the stress response. We identified that the target genes of the MAPK pathway respond to H(2)S; we also identified that a number of transporters respond to H(2)S, these include sugar/carbohydrate transporters, ion transporters, and amino acid transporters. We found many mitochondrial genes to be down regulated upon H(2)S treatment and that H(2)S can reduce mitochondrial oxygen consumption., Conclusion/significance: This study identifies potential molecular targets of the signaling molecule H(2)S in fission yeast and provides clues about the identity of homologues human proteins and will further the understanding of the cellular role of H(2)S in human diseases.